The transcription of eukaryotic genes encoding tissue and developmental stage specific proteins is a complex process involving the interaction of nuclear chromatin with RNA polymerase II, as well as with other transcriptional regulatory factors. The individual members of the globin gene family are temporally regulated to bring about the sequential expression of embryonic, fetal, and adult hemoglobins in the erythroid tissues of many species. The study of these genes has contributed much to our understanding of how selective transcription is accomplished. The epsilon-globin gene is the first of the betalike globin genes to be expressed during human development. To investigate the regulation of this gene we have mapped, in vitro , the sites of interaction between nuclear proteins from erythroid and non-erythroid cells, and DNA sequences in the epsilon-globin promoter. We found that the erythroid factor GATA- 1 bound to three sites in the promoter, and other factors, including SP- 1, bound to the CACCC and CCAAT sites. Using a CAT reporter construct in transient expression assays, we found that the GATA-1 site at -165 is required for transcriptional enhancement by two erythroid specific enhancers. This site has been conserved during the evolution of the mammalian embryonic globin genes. The enhancers each contain their own complement of binding sites for (often the same) nuclear proteins emphasizing the modular nature of transcriptional regulatory regions. In each case the positive-acting regions of the enhancers consisted of AP-1 motifs. Thus, productive promoter-enhancer interactions increasing transcription of the eta-globin gene may require as few as two proteins interacting through two regulatory sites in the DNA.